We have developed a protocol that reproducibly induces transplantation tolerance in individuals undergoing a living related donor kidney transplant. Three subjects have been off all immunosuppression for 10 months, 2 months, and 1 month respectively and have remained chimeric with stable renal function. Two additional chimeric subjects are in various stages of withdrawal from immunosuppression. The goal of this proposal is to expand this protocol to individuals who have already been transplanted and have a living donor that could subsequently provide a bone marrow stem cell product for their recipient (delayed tolerance). This work is important because solid organ, vascularized composite tissue, and islet transplantation currently require nonspecific immunosuppressive agents indefinitely for graft maintenance. These agents have significant toxicities, including nephrotoxicity, opportunistic infections, increased rate of malignancy, diabetes, and hypertension. It has been known for over 50 years that bone marrow chimerism induces tolerance to transplanted organs, cells, and tissues. However, the requirement for close HLA matching and toxicity of ablative bone marrow transplantation has limited the widespread application of this approach. The induction of donor-specific tolerance through durable chimerism in mismatched recipients while avoiding graft-versus-host disease (GVHD) would impact organ and tissue recipients as well as for individuals with hemoglobinopathies, inherited metabolic disorders, and autoimmune disorders. We have strong preliminary data in 8 living donor kidney hematopoietic stem cell transplant (HSCT) recipients, 1 metachromatic leukodystrophy recipient, and 1 sickle cell disease transplant recipient that we can safely establish durable chimerism in highly mismatched donor/recipient pairs. We have successfully established high levels of donor chimerism without GVHD in up to 1/6 HLA matched unrelated donor/recipient pairs. Seven of the eight renal transplant recipients were transplanted with cryopreserved G-CSF mobilized HSCT processed to remove GVHD-producing cells and retain tolerogenic CD8+/TCR- graft facilitating cells (the FCRx). Unfortunately, this successful simultaneous stem cell/kidney approach does not address individuals who have already received a transplanted organ and who have a living donor willing to donate bone marrow. This proposal modifies this approach to accommodate induction of delayed tolerance in a phase I/II protocol. The findings from this study will directly translate to deceased donor organ transplantation. This is of high priority because most organ donors are deceased. Therefore, this novel approach to expand the FCRx technology to delayed tolerance will represent a major milestone in potential commercialization. PUBLIC HEALTH RELEVANCE: Dependence upon anti-rejection drugs in clinical transplantation is expensive and results in significant morbidity and limitation in graft survival. Donor specific transplant tolerance would eliminate the need for drug- based immunosuppression, but has been an elusive goal in the clinic for over a half century. We have developed a potentially transformative approach to achieving transplant tolerance in solid organ transplant recipients based upon a rationally bioengineered stem cell product, termed FCRx. This proposal will determine the safety and efficacy of FCRx for delayed tolerance induction in kidney transplant recipients and will also be directly applicable also to deceased donor organ allograft recipients which comprise the majority of organ transplants.